Analysis of the in vitro transcriptional response of human pharyngeal epithelial cells to adherent pneumococci
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE8527 | GEO | 2007/10/01
SECONDARY ACCESSION(S): PRJNA101655
REPOSITORIES: GEO
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Project description:Infection of the human host by Streptococcus pneumoniae begins with colonization of the nasopharynx, which is mediated by adherence of bacteria to respiratory epithelium. Several studies have indicated an important role for the pneumococcal capsule in this process. Here, we used microarrays to characterize the in vitro transcriptional response of human nasopharyngeal epithelial Detroit 562 cells to adherence of serotype 2-encapsulated strain D39, serotype 19F-encapsulated strain G54, serotype 4-encapsulated strain TIGR4, and their nonencapsulated derivatives (delta-cps). In total, 322 genes were found to be upregulated in response to adherent pneumococci. Twenty-two genes were commonly induced, including those encoding several cytokines (e.g., IL-1-beta, IL-6), chemokines (e.g., IL-8, CXCL1/2), and transcriptional regulators (e.g., FOS), consistent with an innate immune response mediated by Toll-like receptor signaling. Interestingly, 85% of genes was induced specifically by one or more encapsulated strains, suggestive of a capsule-dependent response. Importantly, purified capsular polysaccharides alone had no effect. Over a third of these loci encoded products predicted to be involved in transcriptional regulation and signal transduction, in particular MAPK signaling pathways. Real-time PCR of a subset of ten genes confirmed microarray data and showed a time-dependent upregulation of especially innate immunity genes. Downregulation of epithelial genes was most pronounced upon adherent D39delta-cps, as 68% of the 161 genes identified was only repressed using this nonencapsulated strain. In conclusion, we identified a subset of host genes specifically induced by encapsulated strains during in vitro adherence, and have demonstrated the complexity of interactions occurring during the initial stages of pneumococcal infection. Experiment Overall Design: We used three different pneumococcal strains and their isogenic nonencapsulated derivatives (delta-cps): serotype 2-encapsulated strain D39, serotype 19F-encapsulated strain G54, and serotype 4-encapsulated strain TIGR4. All experiments were performed in triplicate (3 independent biological replicates) and compared to uninfected control Detroit 562 cells. Bacteria were allowed to adhere to the epithelial cells for 2 hours, after which the transcriptional response of the Detroit cells was analyzed using Affymetrix Human U133 Plus GeneChips. In addition to the 6 strains mentioned above, we included transcriptional analysis of the epithelial cell response to low-dose D39delta-cps (giving adherence equivalent to wild-type D39) and purified type 2 capsular polysaccharides.
2008-06-16 | E-GEOD-8527 | biostudies-arrayexpress
Project description:BACKGROUND: Streptococcus pneumoniae, the pneumococcus, is the main etiological agent of pneumonia. Pneumococcal infection is initiated by bacterial adherence to lung epithelial cells. The exact transcriptional changes occurring in both host and microbe during infection are unknown. Here, we developed a time-resolved infection model of human lung alveolar epithelial cells by S. pneumoniae and assess the resulting transcriptome changes in both organisms simultaneously by using dual RNA-seq. RESULTS: Functional analysis of the time-resolved dual RNA-seq data identifies several features of pneumococcal infection. For instance, we show that the glutathione-dependent reactive oxygen detoxification pathway in epithelial cells is activated by reactive oxygen species produced by S. pneumoniae. Addition of the antioxidant resveratrol during infection abates this response. At the same time, pneumococci activate the competence regulon during co-incubation with lung epithelial cells. By comparing transcriptional changes between wild-type encapsulated and mutant unencapsulated pneumococci, we demonstrate that adherent pneumococci, but not free-floating bacteria, repress innate immune responses in epithelial cells including expression of the chemokine IL-8 and the production of antimicrobial peptides. We also show that pneumococci activate several sugar transporters in response to adherence to epithelial cells and demonstrate that this activation depends on host-derived mucins. CONCLUSIONS: We provide a dual-transcriptomics overview of early pneumococcal infection in a time-resolved manner, providing new insights into host-microbe interactions. To allow easy access to the data by the community, a web-based platform was developed ( http://dualrnaseq.molgenrug.nl ). Further database exploration may expand our understanding of epithelial-pneumococcal interaction, leading to novel antimicrobial strategies.
2016-08-26 | GSE79595 | GEO
Project description:We developed spotted oligonucleotide arrays of the S. pyogenes SF370 (an M1 serotype) genome and compared the transcriptomes of streptococci that adhere to Detroit 562 human pharyngeal cells to non-adherent (“associated”) streptococci within the same experiment. We replicated experiments independently and used dye-swaps to incorporate biological and technical variation, biological replicate experiments. Following filtering and normalization, we analyzed data from four biological replicates with robust summary statistics, Bayesian statistics and permutation algorithms to identify genes differentially expressed with significance during pharyngeal cell adherence. This analysis identified 79 genes (4% of the genome) exhibiting statistically significant fold changes in expression (PF value < 0.05) during adherence from 1769 open reading frames represented on the array. The genes encoding several known virulence factors (e.g. cysteine protease SpeB and streptolysin O) were differentially expressed with significance; however, many were either never before associated with adherence (e.g. phage-encoded genes including speH), or of undefined function. Genes demonstrating up-regulation (n= 45) and down-regulation (n= 34) included virulence factors, prophage-encoded transcripts, metabolic genes and transcriptional regulators (Table 2). Undefined or hypothetical genes comprised 27% of differentially expressed genes (n= 21; 11 chromosomally-encoded, 10 phage-encoded). We conducted TaqMan (qRT-PCR) analysis of 11 differentially expressed genes to validate selected microarray hybridization results. We further analyzed the data with neighbor clustering, using a set of novel computational algorithms (termed “GenomeCrawler”) for evaluating bacterial microarray data, which couples the expression profiles of genes with their physical location on the chromosome. When applied to the microarray data from this study, neighbor clustering identified a greater number of differentially expressed genes, which facilitated the reconstruction of known multimeric proteins, complete metabolic pathways and intact virulence loci that would not have been possible without its application. These data sets and algorithms are freely available for download at www.rockefeller.edu/vaf. Keywords: Pharyngeal cell adherent vs. associated
2007-04-27 | GSE7620 | GEO
Project description:Background. Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity. Methods. A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. Results. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements. Conclusions. NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.
2014-11-24 | GSE58329 | GEO
Project description:Analysis of the pulmonary gene expression in two mouse strains BALB/cOlaHsd (BALB/c) and CBA/CaOlaHsd (CBA/Ca) after infection with various serotypes of Streptococcus pneumoniae. BALB/c mice show high resistance to infection with S. pneumoniae strain D39 (serotype 2), while CBA/Ca mice are highly susceptible. The lung samples of BALB/c and CBA/Ca were collected at 6h post-infection with one of the tested pneumococcal serotypes (2, 3, 6B and 19F) and for control animals (PBS-treated). Additionally lung samples from both mouse strains were collected at 12h and 24h post-infection with pneumococcal strain D39. The lists of differentially expressed genes were created by the comparison of infected versus PBS-treated animals and infected BALB/c versus infected CBA/Ca for each pneumococcal strain. The tested hypotheses were: 1) infection with S. pneumoniae will change the pulmonary transcriptomes of both mouse strains 2) The pulmonary gene expression will be specific for mouse strains and for the pneumococcal serotype and 3) The change in the pulmonary gene expression will associate with future clinical outcome of infection or with the type of observed inflammatory responses.
2015-10-28 | GSE61459 | GEO
Project description:RNases perform indispensable functions in regulating gene expression in many bacterial pathogens by processing and/or degrading RNAs. Despite the pivotal role of RNases in regulating bacterial virulence factors, the functions of RNases have not yet been studied in the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus). Here, we sought to determine the impact of two conserved RNases, the endoribonuclease RNase Y and exoribonuclease polynucleotide phosphorylase (PNPase), on the physiology and virulence of S. pneumoniae serotype 2 strain D39. We report that RNase Y and PNPase are essential for pneumococcal pathogenesis as both deletion mutants showed strong attenuation of virulence in murine models of invasive pneumonia. Genome-wide transcriptomic analysis revealed that nearly 200 mRNA transcripts were significantly up-regulated, whereas the abundance of several pneumococcal sRNAs, including the Ccn (CiaR Controlled Noncoding RNA) sRNAs, were altered in the ∆rny mutant relative to the wild-type strain. Additionally, lack of RNase Y resulted in pleiotropic phenotypes that included defects in pneumococcal cell morphology and growth in vitro. In contrast, Dpnp mutants showed no growth defect in vitro, but differentially expressed a total of 40 transcripts including the tryptophan biosynthesis operon genes and numerous 5’-cis-acting regulatory RNAs, a majority of which were previously shown to impact pneumococcal disease progression in mice using the serotype 4 strain TIGR4. Altogether our data suggest that RNase Y exerts a global impact on pneumococcal physiology, while PNPase-mediates virulence phenotypes, likely through sRNA regulation.
2021-09-02 | GSE173392 | GEO
Project description:We used a combination of adherence assays, mutagenesis and functional genomics to identify novel factors involved in adherence. This work identifies a list of novel potential pneumococcal adherence determinants.
2013-09-30 | GSE44947 | GEO
Project description:Control of Streptococcus pneumoniae colonisation at human mucosal surfaces is critical to reducing the burden of pneumonia and invasive disease, interrupting onward transmission, and in achieving herd protection. We hypothesised that the pattern of pneumococcal-epithelial engagement dictates the inflammatory response to colonisation, and that this epithelial sensing is linked to bacterial clearance. Here we have used nasal curette biopsies from a serotype 6B Experimental Human Pneumococcal Carriage Model (EHPC) to visualize S. pneumoniae colonisation and relate these interactions to epithelial surface marker expression and transcriptomic profile upregulation. We have used a Detroit 562 cell co-culture model to further understand these processes and develop an integrated epithelial transcriptomic module to interrogate gene expression in the EHPC model. We have shown for the first time that pneumococcal colonisation in humans is characterised by microcolony formation at the epithelial surface, microinvasion, cell junction protein association, epithelial sensing, and both epithelial endocytosis and paracellular transmigration. Comparisons with other clinical strains in vitro has revealed that the degree of pneumococcal epithelial surface adherence and microinvasion determines the host cell surface marker expression (ICAM-1 and CD107), cytokine production (IL-6, IL-8 and ICAM-1) and the transcriptomic response. In the context of retained barrier function, epithelial microinvasion is associated with the upregulation of a wide range of epithelial innate signalling and regulatory pathways, inflammatory mediators, adhesion molecules, cellular metabolism and stress response genes. The prominence of epithelial TLR4R signalling pathways implicates pneumolysin, a key virulence factor, but although pneumolysin gene deletion partially ameliorates the inflammatory transcriptional response in vitro, critical inflammatory pathways persist in association with enhanced epithelial adhesion and microinvasion. Importantly, the pattern of the host-bacterial interaction seen with the 6B strain in vitro is also reflected in the EHPC model, with evidence of microinvasion and a relatively silent epithelial transcriptomic profile that becomes most prominent around the time of bacterial clearance. Together these data suggest that epithelial sensing of the pneumococcus during colonisation in humans is enhanced by microinvasion, resulting in innate epithelial responses that are associated with bacterial clearance.
2019-01-12 | GSE124949 | GEO
Project description:Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen’s adherence to HeLa cells (39). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we experimentally inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue culture monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. As expected, the cadA mutant did not possess lysine decarboxylation activity and was hyper-adherent to tissue-culture cells. Adherence of the cadA mutant was nearly 2-fold greater than that of the wt and complementation of the cadA defect reduced adherence back to wt levels. Furthermore, the cadA mutant affected the expression of intimin protein. Disruption of the eae gene (encoding the intimin protein) in the cadA mutant significantly reduced its adherence to tissue-culture cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1332 genes was down-regulated and 132 genes up-regulated in the mutant compared to the wild type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins: flagella and F9 fimbriae were up-regulated in the cadA mutant, suggestive of their association with adherence in absence of the Cad regulatory mechanism. Remarkably, in the infant rabbit model, the cadA mutant out-competed the wild type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.
2008-09-18 | GSE11927 | GEO
Project description:Invasive pneumococcal disease is preceded by asymptomatic colonization of the human nasopharynx by Streptococcus pneumoniae. Progression from colonization to invasion is a watershed in the host-pathogen interaction, and exposes the pneumococcus to markedly different microenvironments. This in turn, requires alterations in gene expression profile to adapt to the new niche. One apparent adaptive mechanism is reversible phase variation between “transparent” and “opaque” colony opacity phenotypes. Transparent phase variants colonize the nasopharynx more efficiently than opaque variants of the same strain, while opaque variants exhibit higher systemic virulence. Previous studies have reported quantitative differences in surface components such as the capsule, teichoic acid and certain surface proteins between the two phenotypes, but the underlying regulatory mechanism is not understood. In the present study, we found no differences in expression of key surface proteins between opaque and transparent variants of S. pneumoniae strain D39, but opaque cells produced five-fold more capsular polysaccharide. Subsequent microarray and real-time RT-PCR analysis showed no differences in capsule gene expression, but several genes involved in uridine monophosphate (UMP) biosynthesis were up-regulated in the opaque phenotype. This correlated with significant increases in the intracellular concentrations of both UMP and UDP-glucose, which are essential precursors for capsule biosynthesis. Our data suggest a novel mechanism for pneumococcal capsule regulation, in which rate-limiting precursor pathways are modulated rather than the capsule biosynthetic genes themselves. Keywords: Phase variants
2007-08-17 | GSE8797 | GEO